Not Applicable
1. Field of the Invention
This invention pertains generally to automated imaging and data acquisition systems, and more particularly to a table-top system that automatically quantifies the deposition and aggregation kinetics of sub-micrometer- to sub-millimeter-sized particles at an arbitrary surface by simultaneously determining their spatial and temporal distributions at the surface and their diffusion coefficient, velocity, and concentration distributions in the bulk suspension.
2. Description of the Background Art
Colloids are minute particles that are dispersed in different continuous phases. They range from airborne dust and smoke, to foams and emulsions, to proteins and biological cells. Understanding the behavior of these tiny particles is important to research ranging from drug development to environmental remediation. The deposition kinetics of colloids onto different interfaces (e.g., solid/liquid or gas/liquid interfaces) is key to a variety of their applications. For example, in medical drugs, it affects how colloids disperse corrective proteins in body tissue. On the assembly line, it affects how well the paint sprayed onto car bodies will adhere. In the mining industry, it affects how efficiently valuable minerals (like gold) can be separated from waste rock by froth flotation techniques.
Quantifying the deposition kinetics of colloids requires determining their spatial and temporal distribution at a surface (a liquid/solid or gas/liquid interface); determining the position of each colloid when it arrives at, attaches to, and detaches from that surface; and determining the average concentration (or flux) of colliding, attaching, and detaching particles. The large number of particles (typically thousands) that must be tracked and the time-dependence and randomness of their movements rule out quantifying them by manual means.
Experimental methods used to study colloid deposition in different geometries can be classified into either direct or indirect methods. Indirect methods measure quantities that are unequivocally related to the number of deposited colloids. Examples include: (a) the depletion method, in which the change in the number of colloids in the bulk suspension is measured over time and correlated to the average number of deposited colloids, (b) the packed-bed column method, in which the capture of colloids by the packed-bed of a well-defined granular material is determined, by measuring the difference between the inlet and outlet colloid bulk concentrations, and related to the number of deposited colloids onto the surfaces of the granular material, (c) the light scattering method, in which the intensity of light scattering by deposited colloids, in the direction normal to the incident light beam, is measured and related to the number of deposited colloids, and (d) the radioactive tracer method, in which the radiation emitted by deposited colloids tagged with a suitable xcex3-ray-emitting material is measured and related to their surface concentration.
An inherent limitation to all indirect methods is the inability to quantify the attachment and detachment events of the colloids during the deposition process or their spatial and temporal distribution on the surface. The first two methods (a and b) are also labor intensive and subject to human errors in measuring the changes in the bulk concentration of the colloids. Those changes are typically measured by manually sampling and measuring using, for example, the Coulter principle or light obscuration methods. Moreover, these two methods implicitly assume that the colloid deposition onto the surface is uniform and that the number of deposited colloids equals those lost from the bulk suspension. Thus colloids removed from the bulk suspension by processes other than deposition (e.g., mechanical filtration, coagulation) or by deposition in the sampling tubes are not accounted for, which can potentially skew the experimental results. The last two methods (b and c) are also subject to counting errors due to the background light scattering or radioactivity. Neither method can distinguish between mobile colloids close to the surface and colloids deposited onto the surface.
These limitations of indirect methods have precluded their use in studies aiming at gaining a quantitative understanding of the underlying processes or validating colloid deposition theories or models. Detailed information on the deposition of colloids, larger than xcx9c0.3-0.5 xcexcm, can be obtained in situ using direct visualization and appropriate image processing methods. These methods can track the time when and the position where each particle attaches to and detaches from the surface, thus the temporal and spatial distributions of the particles at the surface. Direct in situ video microscopic techniques have been successfully employed to investigate colloid deposition at the stagnation point, and in parallel-plate channels. These investigations have demonstrated that direct methods are capable of providing important information beyond the capabilities of indirect methods.
Almost all experiments reported on colloid deposition in parallel-plate geometry have employed in situ direct video microscopic techniques. For example, phase-contrast light microscopy has been used in conjunction with an image processing and data extraction routine to investigate the deposition of 736 nm and 830 nm polystyrene colloids from a flowing suspension through a 0.06 cm aperture parallel-plate channel onto glass and plastic substrates. However, because that system has a relatively low spatial optical resolution, an ultralong working distance objective lens with a relatively high magnification is used, reducing the field of view to only xcx9c1.7xc3x9710xe2x88x924 cm2, and hence decreasing the statistical accuracy of the measurements. Further, the image processing and data extraction routine used could not establish full connectivity between deposited colloids in successive images. Also, the number of attached and detached colloids at the surface was determined by labeling the colloids with different gray-scale values. The 8-bit gray-scale used therewith indicates that the number of labeled colloids is limited to a total of 256, thus the data extraction ceased once the total number of attachment or detachment events reaches 256.
The evanescent field technique has also been utilized in conjunction with an elaborate data extraction routine to quantify the deposition of 310-nm diameter fluorescent polystyrene colloids onto the base surface of a rectangular optical glass prism in contact with a flowing colloid suspension in parallel-plate geometry. However, an optical glass prism is necessary to generate the evanescent field at its base surface, which is used as the deposition surface. The underlying parallel-plate channel has an aperture of 0.1 cm. Although it has an optical resolution of xcx9c1 xcexcm, that system possesses some inherent disadvantages, including: (a) because of the discontinuous illumination of the surface, continuous visualization is hard to achieve, limiting the time resolution between measurements, (b) colloid deposition can only be studied at the base surface of the optical glass prism and the colloids need to be fluorescence-tagged to enhance visualization and image contrast, and (c) the laser beam used to illuminate the deposition surface may induce unfavorable heat and radiation pressure, thus affecting the accuracy of the results.
In summary, the direct in situ methods can separate and accurately quantify the attachment and detachment processes of colloids mingled with their deposition onto a surface. They allow experimentalists to verify measured quantities and immediately detect and correct setup errors during the course of an experiment, avoiding costly reruns. These methods, however, have several limitations such as low spatial resolutions ( greater than 1 xcexcm), limited time resolutions (xcx9c2 to 20 minutes), and small fields of view ( greater than 0.035 mm2), in addition to being applicable only to colloids larger than xcx9c0.3-0.5 xcexcm in size. Also, depending on the type of microscopy employed, unavoidable thermal and radiation pressure effects may influence the colloid deposition process. Several uncertainties can also arise from the employed image processing and data extraction methodologies. A major uncertainty in the measurements is caused by the lack of or poor connectivity between colloids in subsequent images. Due to the unavoidable shifting in the view field, a stationary colloid in one image may not correspond to the same colloid in subsequent or preceding images. Another source of uncertainty in the measurements by the direct methods is missing some attachment and detachment events taking place during the xe2x80x9cblindxe2x80x9d time interval between images. Thus, the measured attachment and detachment concentrations and fluxes may be substantially less than the actual values.
Currently, there is no commercially available technology with the functionality needed to quantify the deposition and aggregation kinetics of minute particles at an arbitrary surface. Software is available, such as the xe2x80x9cMSQ Materials Analysis Systemxe2x80x9d (Definitive Imaging, Ltd.), that runs on an Optimas image processor and is designed to be interfaced to visualization hardware. However, MSQ obtains data such as grain size, sphericity, and volume fraction from only nontransient images and cannot quantify attachment/detachment events. Additionally, commercially available instruments are limited in their measurement capabilities and are costly. Examples are the DELSA 440SX (available from Beckman Coulter, Inc.) which measures zeta potential and particle size, and the Multisizer 3 which measures particle size and concentration (also available from Beckman Coulter, Inc.). There are also research instruments that have limited functionality.
Therefore, there is a need for an automated system that offers greater measurement accuracy, simultaneously captures and analyzes information not only on particles deposited at the surface but also on particles moving close to the surface, has a larger field of view so as to provide better statistical data, is suitable for imaging and analyzing submicrometer-sized particles down to xcx9c200-nm-sized particles, can quantify particle deposition onto relatively rough surfaces such as those of rock and metals, has full connectivity, and produces more data for improved measurement accuracy. The present invention satisfies those needs, as well as others, and overcomes deficiencies found in conventional imaging and data acquisition systems.
The present invention comprises a video-microscopic imaging and data acquisition system that makes the study of colloid deposition and aggregation kinetics simple, cost-effective, fast, and consistent. In general terms, the invention visualizes and characterizes colloidal particles that are suspended in or at the surface of a parallel-plate test cell. The colloids appear as bright specks against a dark background. In a typical experiment, the invention acquires, processes, and analyzes over 10,000 images, extracting from them the data needed to fully quantify colloidal deposition kinetics. Typical output data includes (i) the cumulative concentration of colloids attaching to and detaching from the test cell""s surface, (ii) the evolution of mobile and immobile colloids at the cell""s surface, (iii) and the concentration profile of colloids across the bulk suspension. Key data are extracted and displayed in real time, enabling the user to quickly identify and correct any experimental setup errors.
The present invention enables a person to quantify colloidal processes in real time over periods of up to several days. It combines commercial optical and data-processing equipment with custom software to automatically acquire, store, digitize, process, enhance, and analyze thousands of analog images of colloids at an interface and in suspension. The invention acquires up to thirty images per second and from them extracts the information needed to fully quantify colloidal deposition and to measure the colloids"" concentration, diffusion coefficient, and velocity in the suspension. In principle, the invention can also measure their zeta-potential distributions in the bulk suspension.
By way of example, and not of limitation, the invention employs a modified dark-field microscopy to visualize colloidal particles. A suspension of colloids is placed in a parallel-plate test cell approximately 200 micrometers in aperture. The cell is illuminated from below by a 300-watt halogen bulb. Before illuminating the cell, light from the bulb passes through an iris diaphragm and a dry dark-field condenser. The condenser has a numerical aperture of 0.8-0.9, and the iris diaphragm passes only the minimum light intensity needed to illuminate the suspension. Thus only low-numerical-aperture light scattered from the colloids enters the objective lens of a light microscope placed above the test cell.
By adjusting the angle of incident light and correcting for optical aberrations, high contrast from the suspended colloids is achieved even though their size is far below the microscope""s resolution limit. The microscope""s relatively low magnification (only 20xc3x97) produces a large field of view (xcx9c0.082 mm2) and offers a reasonable compromise between image resolution and contrast. The colloids appear as bright specks against a dark background (see image), with diffraction making them appear much larger than actual size. A correction cap on the objective lens allows the system to focus on different layers of colloids throughout the suspension. A 3.3xc3x97 photo eyepiece installed within the microscope""s phototube projects the image formed to a charge-coupled device (CCD) camera, whose analog output is then digitized and processed with custom application-specific software. Written in Analytical Language for Images (ALI), the software automates data acquisition, processes and analyzes the sequence of images obtained, and extracts real-time data for graphical display.
The software comprises a main routine and three subroutines. The main routine automates an experiment and applies two algorithms: one captures the kinetics of colloidal deposition onto the surface, and the other determines selected colloidal suspension properties. After processing the acquired images, the main routine extracts the evolution of mobile and immobile colloids at the test cell""s upper or lower surface, the cumulative concentration over time of colloids attaching to and detaching from that surface, and the concentration profile of colloids across the suspension. The main routine also saves three sequences of images: one of both mobile and immobile colloids at the surface, one of immobile colloids at the surface, and one of colloids in different layers across the suspension.
The subroutines enable the system to distinguish between colloids that arrive at the surface, collide with it, and attach to it, quantifying the concentration and rate of each set. The subroutines also provide detailed spatial and temporal distribution data on colloids at the surface. When combined, this data allows for quantification of the colloidal deposition process.
The subroutines also solve two major problems encountered in analyzing colloidal deposition data. The first involves determining the concentrations of colloids colliding with and permanently attaching to the surface; the two quantities strongly depend on the time interval between images, xcex94xcfx84. Experiments performed under the same conditions but with different values of xcex94xcfx84 give substantially different results, precluding their comparison. By superimposing images of the surface obtained at different values of xcex94xcfx84, several curves representing the cumulative concentration of attached particles at selected times for each value of xcex94xcfx84 are created. Then by establishing a mathematical relationship between these curves and xcex94xcfx84, two curves that are independent of the time interval are obtained; one for the concentration of colliding particles, and the other for the concentration of attached particles.
The second problem involves establishing xe2x80x9cconnectivityxe2x80x9d between colloid images taken over long periods of time. Connectivity means that a stationary particle in one image corresponds to one, and only one, particle in later images. In accordance with the present invention, connectivity is ensured by choosing a group of stationary particles as a frame of reference for all images and then selecting one of the particles as the reference point to which the coordinates of all other particles are related. If that particle moves in later images, another particle in the original group is selected as a new reference point.
The present invention is ideal for basic and applied research aimed at quantifying the kinetics of sub-micrometer particles deposited onto varied surfaces or aggregated in varied solutions. Used alone, the invention can provide accurate particle concentration data in two ways: (i) by counting the number of particles in different layers across a suspension, which can be done simultaneously while performing a deposition experiment (with xcx9c10% accuracy), or (ii) by using a solution chemistry that allows particles to adhere to the surfaces of the test cell and then counting them at those surfaces. Used in conjunction with an electrophoresis cell, the invention can also be used to study the zeta potential of particles by automatically determining the particles"" mobility in the direction normal to an applied electric field. The zeta potential of particles dispersed in aqueous media is an important characteristic that determines their electrostatic interaction with each other and with substrate materials.
An object of the invention is to obtain particle arrival and deposition concentrations independent of the experiment design.
Another object of the invention is to capture both particles immobilized at the surface and those moving close to it, and distinguishes between them, without refocusing.
Another object of the invention is to obtain particle concentration, diffusion coefficient, and velocity across the test cell during deposition or aggregation experiments.
Another object of the invention is to capture, process, and analyze over 10,000 images during the course of a typical experiment.
Another object of the invention is to provide a system wherein users can interact with the system remotely through the Internet and can share real-time data with others during the course of an experiment.
Another object of the invention is to extract and display key data on-line, enabling users to quickly identify and correct any experimental setup errors.
Another object of the invention is to establish connectivity over time periods on the order of days, reducing significant errors that result when connectivity is lacking.
Another object of the invention is to use standard light as an illumination source, which eliminates the thermal effects that can be induced by lasers, reduces equipment and operating costs, and suits the system to biological studies.
Another object of the invention is to provide an imaging system with a wide field of view.
Another object of the invention is to use various materials as the substrate for particle deposition (e.g., thin sections of natural rocks or metals as well as gas/liquid interfaces).
Another object of the invention is to visualize particles at varied depths in a suspension without deterioration so that those particles are analyzed and quantified with the same accuracy as surface particles.
Further objects and advantages of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the invention without placing limitations thereon.